The data here confirm a high degree of variability in viral load results from assays targeting CMV, EBV, and BKV. Compared with results from viral load testing for HIV and HCV, this variability becomes even more apparent. While a limited number of studies have addressed this issue, most have focused solely on lack of standardized quantitative calibrators as a primary reason for differences in precision and accuracy among laboratories (2
). This study is perhaps the first to look comprehensively at multiple factors in the complex process of quantitative PCR. The commercial detection reagents, the calibrator, extraction and detection methods, and the target gene selected can all play a role in magnitude and variability of quantitative results.
That the calibrator selected was shown to affect MVL and variability of CMV results is not surprising. Several studies have now shown that different quantitative standards may have dramatic effects on viral load results (6
). The consistent results seen for HIV and hepatitis viral load proficiency testing can be, at least in part, attributed to the fact that international quantitative standards have been broadly adopted for these targets (8
); such international quantitative standards were not available for CMV, EBV, and BKV at the time of data collection for this study. The recent introduction of a WHO quantitative CMV standard can be expected to help address this issue, and standardization for EBV and BKV is planned (4
The impact of the detection reagent selected is also consistent with that in various reports in the literature. In fact, the reagent used was consistently ranked as one of the highest contributors to overall assay variability, across all three target viruses in this study. While not the subjects of a systematic review, many detection reagents have shown variability in performance characteristics when different reagents were compared (6
). Such differences can be expected to affect both accuracy and precision, as seen in this study. The relatively low number of FDA-approved assays for HIV and hepatitis virus quantification may contribute to the uniformity of results for those targets. This study was not intended as a comparative analysis of commercial detection reagents, and the data shown cannot be used to assess the superiority of one product over another. However, it is clear that not all detection reagents produced equivalent results and that variability of results should be carefully assessed when validating viral load assays for routine clinical practice.
Target gene selection has been the subject of much study. It has previously been shown that target gene selection represents a critical part of the test design process, as polymorphisms vary in frequency depending on genomic region (7
). Assays targeting regions of increased sequence heterogeneity may be expected to show effects on sensitivity and on quantitative results. Consistent with this premise, findings here showed that the choice of genetic amplification target affected the MVL for all three targets. A substantial proportion of the overall quantitative variability seen for all three viruses could be accounted for by this factor. While these analyses were sufficient to indicate the importance of genetic target selection as a contributor to changes in MVL and RV, further inferences cannot be made to explain the behavior of individual targets.
Extraction is often overlooked as a key to assay performance. While findings were limited by the fact that most users now rely on silica-based (column/membrane or magnetic bead) extraction, a substantial minority continue to use liquid-phase extraction. These users reported diminished MVL and variability of results. The former may reflect improved yield, purity (removal of inhibitors), or both in the newer, solid-phase systems. The specific findings here may be less important than is the underlined importance of sample preparatory methods and their potential contribution to assay accuracy and precision.
Factors lacking an apparent association with changes in MVL or variability may be as revealing as those that did show such an association. Whether or not calibrators were extracted and how frequently they were assayed (periodically or with every PCR run) have been the subjects of much debate. The factors go beyond convenience and effects on variability, as recalibration of every PCR run can substantially impact cost and turnaround time for results. Neither calibrator extraction nor frequency of full quantitative calibration was associated with altered MVL or variability. Similarly, and perhaps surprisingly, the use of automated pipetting devices did not afford any clear benefits in terms of uniformity of results, compared to manual pipetting devices. Although such tools may save time and their value in reducing repetitive motion injuries is unquestioned, they did not contribute to reduced result variability in this study.
This study was limited by the number of survey participants for each analyte. CMV, with the largest number of responding laboratories, demonstrated the most factors reaching statistical significance in their effects on MVL and variability of results. Clearly, had larger numbers of participants tested for EBV and BKV (as well as for CMV), the number and strength of our findings may have been improved. Findings with respect to relative variability with the use of different calibrators may have been biased to some extent by the fact that some calibrators were used only with detection reagents produced by the same manufacturer (for example, Roche and Qiagen). Variability and MVL were examined separately in this study, but they are actually integral to one another. Although differences in MVL may be distinguished when variability is equal, major differences in variability between two groups may artifactually give the appearance of discrepant means. While the proficiency testing material was handled in a uniform manner prior to distribution, and for each virus was the product of a single, homogeneous production lot, some variation among aliquots cannot be ruled out. Furthermore, this study was not designed to measure the effects of preextraction manipulation and storage conditions. Clearly, there are factors involved in the process of viral load testing that either were not queried of participants or could not be adequately measured here, as evidenced by a substantial proportion of variability that remained unaccounted for in the analysis. Finally, although survey questions were designed to be as simple and unambiguous as possible, some imprecision or inaccuracies may have been introduced in participant interpretations and responses.
Future efforts in this area may include larger studies, perhaps isolating each of the variables seen to be important here. The contribution of calibration variability to imprecision and inaccuracy of viral load measurements cannot be underestimated, and the introduction of international standards can be expected to have a significant and beneficial impact. Repeating some of this work in the presence of such standards may further enable the identification of other important factors. These may in turn be optimized, leading to an iterative process for improving the accuracy and reproducibility of these important tests.
The findings here build on the work of others in clarifying the sources of nonuniformity in viral load testing by molecular diagnostic methods. The value gained in identifying such factors may suggest ways that individual users, as well as commercial manufacturers, may work toward systems that give the same results no matter when, where, or how many times a sample is tested. Improvement of test precision and accuracy can ensure portability of results, enable patients to be more accurately monitored as they move to different institutions, allow for consistency in the literature, and allow the establishment of more informative thresholds for therapeutic and prognostic purposes.